Readouts of orthogonal cargo localization screen. (A and B) Bar graphs showing readouts generated from the screen with VBC-generated crRNAs. Quantification of (A) localization ratio (perinuclear versus peripheral) and the total number for GFP-BICD2N-FRB and PTS-RFP-FKBP spots in U-2 OS PEX cells treated with rapamycin and (B) localization ratio (perinuclear versus peripheral) of EEA1, TGN46, or LAMP1 spots in unmodified, untreated U-2 OS cells. “(V)” and “(D)” indicate crRNAs synthesized based on the VBC score or from the original “Horizon Discovery” set, respectively. Data points represent mean aggregation from at least three independent experiments (minimum of 100 cells analyzed per well; four wells analyzed per condition). EEA1 ratio values were log-transformed for normal distribution. Bold lettering indicates crRNAs that were novel components of the dynein–dynactin cluster of phenotypic profiles. Error bars signify SD. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparison against NTC). (C) Representative images of LAMP1 staining in U-2 OS cells treated with the indicated crRNAs. Scale bar, 20 µm.